The long-term goal of our research program is to develop an estrogen replacement therapy that meets the unique requirements of the brain by activating estrogen-inducible mechanisms of memory and neuroprotection without activating mechanisms of proliferation within the uterus or breast. To achieve this long-term goal, multiple levels of mechanistic understanding of estrogen receptor function in brain must first be achieved. Towards elucidating mechanisms of estrogen promoted neuroprotection, we propose a model of estrogen-inducible proactive adaptation as a strategy whereby estrogen proactively protects neurons against insults of calcium dysregulation. The proposed model incorporates both novel mitochondria mechanisms of estrogen action and several existing estrogen-inducible pathways into a unified concept of proactive adaptation. Four specific aims are proposed. Specific Aim 1 will determine essential basics and generalizability of 17beta-estradiol (E2)-induced mitochondrial sequestration of calcium. Specific Aim 2 will investigate the impact of 17beta-estradiol on the threshold for mitochondrial Ca 2+ sequestration and the underlying mechanism for the shift in threshold. Specific Aim 3 will address the mechanism by which 17beta-estradiol protects against increased mitochondrial calcium load to prevent mitochondrial dysfunction. Specific Aim 4 will determine the mechanism underlying 17beta-estradiol regulation of Bcl-2 family of proteins. Throughout each of the specific aims, we will determine whether 17beta-estradiol-induced mitochondria mechanisms activated in vitro are present in vivo. Seven technological approaches will be extensively utilized: neuronal culture, fluorescent intracellular calcium imaging, biochemical analyses of enzyme activation, immunocytochemical protein labeling, Western blot, mitochondrial isolation and HPLC for polyamines. Mitochondrial function will be assessed within cultured hippocampal neurons and in mitochondria derived from adult rat hippocampal neurons. Results of the proposed studies will provide a unified mechanistic model of estrogen-induced neuroprotection that incorporates both novel mitochondria mechanisms of estrogen action and estrogen-inducible MAPK, AKT and antiapoptotic pathways. From a clinical perspective, elucidation of the sites and targets of estrogen action should have a clear impact on both the use of estrogen replacement therapy for the prevention of neurodegenerative disease and the future design of target specific estrogens. [unreadable] [unreadable]